Process for the purification of epi-hne proteins

ABSTRACT

The present invention concerns a process for the purification of EPI-HNE proteins, from the culture medium of a host strain for the expression of said proteins, comprising the steps of: (a) passing a derived part of the culture medium over an expanded bed of cationic exchange adsorbent or a mechanically and chemically inert micromembrane, in order to recover an eluate, (b) optionally conducting chromatographic separation of proteins, according to their hydrophobicity, on the resulting eluate, (c) passing the resulting eluate over a cationic exchange column, (d) optionally filtering the resulting medium such as to obtain a sterile filtrate, and optionally further comprising the step of (e) causing precipitation of EPI-HNE in a crystallised form and recovering the protein crystals.

[0001] The present invention concerns a novel process for thepurification of EPI-HNE proteins. International Patent Application WO96/20278 to Ley et al. describes a number of genetically engineerednovel proteins which inhibit human neutrophil elastase (hNE). Asindicated in the above-cited patent application, human neutrophilelastase (also known as human leukocyte elastase) is one of the majorneutral proteases of the azurophil granules of polymorphonuclearleukocytes. This enzyme is involved in the elimination of pathogens, andin connective tissue restructuring.

[0002] The principal systemic inhibitor of hNE is the α-1-proteaseinhibitor, formerly known as α1 antitrypsin. In a certain number ofpathological situations (hereditary disorders, emphysema, cysticfibrosis, Adult Respiratory Distress Syndrome ARDS, Chronic ObstructivePulmonary Disease COPD), this inhibitor is either not present insufficient amounts in the bloodstream or is inactivated, leading touncontrolled elastolytic activity of hNE, which causes extensivedestruction of lung tissue.

[0003] WO 96/20278 thus proposes novel proteins which are stable,non-toxic, highly efficacious inhibitors of hNE. These inhibitors formpart of a group of inhibitors derived from a Kunitz-type inhibitorydomain found in basic pancreatic trypsin inhibitor (BPTI) or a proteinof human origin, namely the light chain of human Inter-α-trypsininhibitor (ITI). They are, inter alia, EPI-HNE-1, EPI-HNE-2, EPI-HNE-3and EPI-HNE-4. The inhibitors of WO 96/20278 are produced bybiotechnological methods and contain modified DNA sequences, withrespect to the biological Kunitz domains, which render them highlypotent. One of these inhibitors, EPI-HNE-4, is of particular interest.

[0004] WO 96/20278 describes preparation of Pichia pastoris productionsystems for hNE inhibitors EPI-HNE1, EPI-HNE-2, EPI-HNE-3 and EPI-HNE-4,protein production and purification (see in particular Examples 10-15).

[0005] Yeast Pichia pastoris mutant strain GS115 containing a nonfunctional histidinol dehydrogenase gene (his4) was transformed byexpression plasmids comprising a sequence encoding the S. cerevisiaemating factor alpha prepro peptide fused directly to the amino terminusof the desired hNE inhibitor, under control of the upstream inducible P.pastoris aox1 gene promoter and the downstream aox1 transcriptiontermination and polyadenylation sequences. The expression plasmids werelinearized by SacI digestion and the linear DNA was incorporated byhomologous recombination into the genome of the P. pastoris strain GS115by spheroplast transformation, selection for growth in the absence ofadded histidine and screening for methanol utilization phenotype,secretion levels and gene dose (estimated by Southern Blot). Strainsestimated to have about four copies of the expression plasmid integratedas a tandem array into the aoxl gene locus were thus selected.

[0006] Cultures of selected strains were first grown in batch mode withglycerol as the carbon source, then following exhaustion of glycerolgrown in glycerol-limited feed mode to further increase cell mass andderepress the aox1 promoter and finally in methanol-limited feed mode.During the latter phase the aox1 promoter was fully active and theprotein was secreted into the culture medium.

[0007] Ley et al. then indicate that the proteins were purified usingstandard biochemical techniques, the specific purification procedurevarying with the specific properties of each protein. Briefly, theculture medium was centrifuged, the supernatant was subjected tomicrofiltration and subsequently to ultrafiltration, possibly todiafiltration, and then the protein was recovered by ammonium sulfateprecipitation and ion exchange chromatography.

[0008] Specific production and purification procedures are described inWO 96/20278 only for EPI-HNE2 and EPI-HNE3, the overall purificationyield starting from a culture medium containing 720 mg/l and 85 mg/lbeing about 30% and 15%, respectively, for a purity for each of thoseproteins of over 95% as assessed by silver-stained high-resolution PAGEanalysis. The purified EPI-HNE3 is disclosed to be substantially free ofa alternatively-processed form of this protein having an amino extensionof 7 to 9 amino acid residues and representing about 15-20% of the hNEinhibitory activity.

[0009] EPI-HNE1 and EPI-HNE4 are only taught as being produced andpurified in a manner analogous to EPI-HNE2 and EPI-HNE3, without anydisclosure of the specific purification procedure, the purificationyield or the purity of the purified protein.

[0010] The procedures for the purification of EPI-HNE proteins disclosedin WO 96/20278 are not suited for obtaining amounts of purified proteinsof acceptable pharmaceutical quality on an industrial scale. Indeed thesucession of centrifugation, microfiltration and ultrafiltration stepscannot be performed in an economically viable manner on a large scaleand leads to insufficient yields. Besides, as shown by the applicant inExample 1, the purified protein is not separated from pharmaceuticallyunacceptable contaminants such as green fluorescent pigments secreted byP. pastoris.

[0011] The problem addressed by the invention is therefore to develop aprocess for the purification of an EPI-hNE protein from the culturemedium of a host strain capable of expressing that protein, which doesnot have the above-mentioned drawbacks.

[0012] The above problem is solved by the invention as defined in theappended claims.

[0013] The process of the invention allows to purify large quantities ofan EPI-HNE protein from the culture medium of a host strain expressingthat protein, with an overall yield of at least about 40%, preferably atleast 60%, to a degree acceptable for pharmaceutical uses.

[0014] The invention concerns a process for the purification of EPI-HNEproteins, from the culture medium of a host strain for the expression ofsaid proteins, comprising the steps of:

[0015] (a) passing a derived part of the culture medium over an expandedbed of cationic exchange adsorbent or a mechanically and chemicallyinert micromembrane, in order to recover an eluate,

[0016] (b) optionally conducting chromatographic separation of proteins,according to their hydrophobicity, on the resulting eluate,

[0017] (c) passing the resulting eluate over a cationic exchange column,

[0018] (d) optionally filtering the resulting medium such as to obtain asterile filtrate

[0019] The host strains used for the expression of the EPI-HNE proteinsmay be selected among suitable strains of any prokaryotic or eukaryoticorganisms transformed by a vector for the expression of said proteins.Preferably, the host strain is selected from the group consisting ofbacteriae such as Escherichia Coli, Streptomyces or Lactococcus andyeast such as Saccharomyces cerevisiae, Schisaosaccharomyces pombe,Yarrovia lipilitica, Hansenula polymorpha, Kluyveromyces lactis, Pichiamethanolica and Pichia pastoris, which are capable of expressing highlevels of a recombinant protein. Even more preferably, the host strainis Pichia pastoris.

[0020] The expression “derived part of the culture medium” has aspecific meaning in the context of the present invention, as indicatedhereafter.

[0021] When the host strain is capable of secreting the protein outsidethe cell, the derived part of the culture medium in step (a) is theculture broth.

[0022] When the host strain expresses the protein inside the cell, e.g.in the periplasm, the derived part of the culture medium in step (a) isthe lysis product thereof.

[0023] Preferably the above process comprises the further step of:

[0024] (e) causing precipitation of EPI-HNE in a crystallised form andrecovering the protein crystals.

[0025] Step (a) is an expanded bed adsorption step conducted on acationic exchange adsorbent, well known in the art (see for example, M.Hansson et al., 1994, Biotechnol. 12, pp. 285-288) or a filtration stepon a mechanically and chemically inert micromembrane.

[0026] The cationic exchange adsorbent may be based on cross-linkedagarose or cross-linked sepharose, modified through the inclusion of aninert core material, such as crystalline quartz, to provide the requiredhigh density for stable bed expansion, preferably from 1.1 to 1.5 g/ml,or zirconium having a higher density between about 3.5 mg/ml, whichallows a higher flow rate, and by attachment of surface cationicfunctional groups, notably strong cationic groups such as sulfonate. Theparticles may be spherical, with a mean particle size of 100-300 μm,preferably from 180-220 μm. A suitable cationic exchange adsorbent isStreamline SP or Streamline SPXL from Amersham-Pharmacia.

[0027] In a preferred embodiment of the invention, the passage over anexpanded bed is performed as follows:

[0028] Ten litres of chromatographic matrix (Streamline SP fromAmersham-Pharmacia) is equilibrated in 50 mM ammonium acetate pH 3.5 andfluidized in the same buffer to 30 l at 300 cm/h. The system allows thedirect loading of the collected culture medium, thus avoiding thefurther steps of centrifugation, microfiltration and ultrafiltration ofthe prior art, and allowing increase in yields. After loading, thecolumn is washed in the 10 mM ammonium acetate pH 3.5 to obtain anabsorption at 280 nm below 0.05. The beads are packed to 10 l andEPI-HNE-4 is recovered by washing the column in 1 M ammonium acetate pH4.5 buffer.

[0029] A mechanically and chemically inert micromembrane here means amicrofiltrating medium which has a wide pH compatibility, preferably atleast between 3 and 14, allows a high flux of filtrate and is able towithstand high burst pressure, preferably at least 10 bars, inparticular at least 50 bars, and backpulsing. Commercially availablesuch membranes are generally made of ceramics. Examples of suchmembranes are Membralox® (J.M. Separations), Carbosep®/Kerasep™ (Rhodia)and ProCell™ Large Process Scale Cartridge (A/G Technology Corporation).

[0030] Optional step (b) is a hydrophobic interaction or reverse phasechromatography step which allows to separate a mixture of proteinsaccording to their hydrophobicity. That step may be useful whenfermentation is performed on a large scale (over 1 cubic meter) andpossibly additional contaminants may be present.

[0031] Hydrophobic interaction or reverse phase chromatography is wellknown in the art and described notably in “Protein purification, Secondedition Principles, High resolution Methods and applications”, Ed. J. C.Janson and. L. Rydén, pp 239-282.

[0032] A hydrophobic interaction chromatography step is preferable sinceit can be performed without using organic solvents.

[0033] A suitable matrix is a polymer such as polystyrene,polystyrene-divinylbenzene crosslinked methacrylate, modifiedmethacrylate, polyacrylamide, crosslinked agarose or crosslinkedsepharose, which carries a ligand having a hydrophobicity between thehydrophobicity conferred by a C2 alkyl group and the hydrophobicityconferred by a C8 alkyl group. Examples of such ligands are ethyl,propyl, isopropyl, butyl, isobutyl, tertiobutyl, pentyl, isopentyl,hexyl, phenyl, isopropanol, isobutanol, C4-C6 ether. Phenyl ispreferred.

[0034] Examples of suitable matrices are Toyopearl hexyl 650C (modifiedmethacrylate with hexyl ligand), Toyopearl ether 650M (modifiedmethacrylate with C5-ether ligand), Toyopearl butyl 650M (modifiedmethacrylate with butyl ligand) and Toyopearl phenyl 650M (modifiedmethacrylate with phenyl ligand), available from Tosohaas; Resource ETH(polystyrene-divinylbenzene with ethyl ligand), Resource ISO(polystyrene-divinylbenzene with isopropanol ligand), Resource Phe(polystyrene-divinylbenzene with phenyl ligand), Phenyl-sepharose FastFlow (crosslinked sepharose with phenyl ligand), available fromAmersham-Pharmacia; Macroprep t-butyl (crosslinked methacrylate witht-butyl ligand) and Macroprep Phenyl (crosslinked methacrylate withphenyl ligand) available from Bio-Rad.

[0035] Most preferably the matrix is Phenyl-sepharose Fast Flow fromAmersham-Pharmacia (rigid matrix of 45-165 μm beads of crosslinkedsepharose with phenyl ligand).

[0036] Where step (b) is a reverse phase chromatography step a suitablematrix is a polymer such as polystyrene, polystyrene-divinylbenzene,crosslinked methacrylate, modified methacrylate, which carries no ligandor a ligand having a hydrophobicity greater than that conferred by a C10alkyl group.

[0037] Examples of suitable matrices are CG300-M(polystyrene-divinylbenzene), CG161-M (polystyrene-divinylbenzene),available from Tosohaas; Resource 15RP (polystyrene-divinylbenzene),Resource RPC (polystyrene-divinylbenzene), available fromAmersham-Pharmacia; Macroprep High S C18 (crosslinked methacrylate withC18 ligand) available from Bio-Rad; Poros 50R1 et Poros 50R2(polystyrene-divinylbenzene), available from Perseptive Biosystems.

[0038] In a preferred embodiment of the process according to theinvention, the matrix upon which the reverse phase chromatography isconducted is constituted by synthetic hydrophobic beads. Preferably, thematrix is a polystyrene-divinylbenzene matrix, preferably with beads of60-90 μm, more preferably 75 μm, and with a pore size greater than 150Å, preferably a pore size of 300 Å.

[0039] Most preferably, the matrix is a CG 300 M matrix, available fromTosoHaas (polystyrene matrix, 75 μm beads with average pore size of 300Å).

[0040] In the process according to the present invention the maincontaminants after the expanded bed chromatography or microfiltration,are the pigments from the host cells, which include polyphenols anddegradation products thereof.

[0041] The cationic exchange column of step (c) is selected such thatthe EPI-HNE protein does not precipitate while passing through thecolumn. In order to obtain a high yield, this step is preferablyconducted at a pH between 1.8 and 5.0, preferably between 2.0 and 3.0.The elution is performed under conditions such as to obtain a highconcentration of EPI-HNE (over 10 mg/ml) and low ionic strength (lessthan 25 mS/cm). Such an elution might be obtained by “displacementchromatography” using a polycation such as polyethyleneimine,polylysine, polyargininine or any other polymer, such as e.g.polyvinylpyrrolidone (PVP), which contains a high density of positivecharges at a pH between 1.8 and 5. The same result may be obtained byloading a diluted peak after step (a) or (b) on a weak cation exchangerat a pH between 4.5 and 5.0 and by elution at pH 2.0 since the weakcation exchanger possess no anionic charge at such acidic pH andretained no more cationic proteins.

[0042] Examples of a suitable matrix for the cationic exchange columninclude Macroprep High S matrix (rigid matrix based on cross-linkedmethacrylate carrying sulphonate surface groups) from BioRad; S.Sepharose Fast Flow (crosslinked agarose carrying sulphonate groups) andCarboxymethyl Sepharose (crosslinked sepharose carrying carboxymethylgroups) from Amersham-Pharmacia.

[0043] Step (c) is a conventional cationic exchange step which allows toconcentrate the EPI-HNE protein to allow its high yield and high speedcrystallisation if step (e) is used and, if an organic solvent is usedduring step (b), to eliminate traces thereof which are not acceptablefor a pharmaceutical.

[0044] The aim of optional step (d) is to eliminate all microorganismssuch as e.g. bacteriae or yeast. This step is of particular importancewhen the conditions subsequently used are capable of causing substantialgrowth of microorganisms, e.g. when step (e) is performed over a timeperiod over 3 hours. The filtration under sterile conditions isperformed under conditions well known in the art, for example at 0.22μm.

[0045] In step (e) the precipitation of EPI-HNE in a crystallised formis suitably performed in an aqueous vehicle at a pH comprised between3.0 and 8.0, preferably 4.0 and 6.0, most preferably at a pH of 4.0 to5.0, the concentration of the EPI-hNE protein in the aqueous vehiclebeing comprised between 1 and 80 mg/ml, preferably between 2 and 50mg/ml.

[0046] Step (e) is preferably performed using cycles ofcrystallisation/sonication. The latter indeed dramatically increase thekinetics of precipitation, thereby substantially reducing the timenecessary for carrying out step (e).

[0047] The aqueous vehicle is suitably a saline solution having aniso-osmotic pressure. The saline solution may comprise sodium orammonium acetate and sodium or ammonium chloride, or sodium or ammoniumcitrate.

[0048] The term “crystallised form” of EPI-HNE here means an insolubleform of that protein having a rod-like structure and a size mainly below10 μm.

[0049] The crystals of EPI-HNE are recovered by centrifugation orfitration.

[0050] The recovered crystals may be directly suspended in apharmaceutically acceptable buffer or freeze-dried in a suitable buffersuch as ammonium bicarbonate 10 μM or spray dried into an aerosolpowder.

[0051] The EPI-HNE proteins to be purified according to the process ofthe present invention may be selected from the group consisting ofEPI-HNE-1, EPI-HNE-2, EPI-HNE-3, and EPI-HNE-4. EPI-HNE-4 is preferred.

[0052] The invention will be illustrated by the following examples.

EXAMPLE 1 Purification of EPI-HNE4 Using a Slight Modification of theMethod Disclosed for Purifying EPI-HNE3 in Example 13 of WO 96/20278(Comparative Example)

[0053] Yeast Production System.

[0054] The hNE inhibitors are produced as secreted proteins in theculture supernatants of high cell density Pichia pastoris strain GS115fermentations. Expression plasmids are constructed by ligating syntheticDNA sequences encoding the Saccharomyces cerevisiae mating factor CLprepropeptide directly to the 5′-terminus of synthetic DNA encoding thedesired hNE inhibitor. This fusion gene is sandwiched between anupstream inducible P. pastoris aox1 gene promoter and downstream aox1gene transcription termination and polyadenylation sequences that arecarried on a plasmid that also encodes a S. cerevisiae his4 gene.Linearized expression-plasmid DNA is incorporated by homologousrecombination into the genome of the P. pastoris strain GS115 byspheroplast transformation. Regenerated spheroplasts are selected forgrowth in the absence of added histidine. Individual isolates arescreened for methanol utilization phenotype (mut +), secretion levels,and gene copy number. Strain PEY-53 secreting a high level of EPI-HNE-4was thus selected. This strain is estimated by Southern analysis ofgenomic DNA to contain four copies of expression plasmid DNA integratedinto the aox1 gene locus.

[0055] Protein Production

[0056]P. pastoris strain PEY-53 are grown in mixed-feed fermentationssimilar to the procedure described in WO 96/20278, with the differencethat pressurized air is used instead of purified oxygen. Briefly,cultures are first grown in batch mode with glycerol as the carbonsource. After exhaustion of glycerol, the cultures are grown for aboutfour hours in glycerol-limited feed mode to further increase cell massand to derepress the aox1 promoter. In the final production phase, thecultures are grown in methanol-limited feed mode. During this phase, theaox1 promoter is fully active and the hNE inhibitors are secreted intothe conditioned medium (C.M.) The final concentration of EPI-HNE-4 inthe PEY-53 fermentation C.M. was about 600 mg/L as determined bySDS-PAGE analysis.

[0057] The major molecular species produced by PEY-53 cultures is theproperly processed EPI-HNE-4 protein. However, this strain also secretesabout 5-20% of an alternatively-processed protein having slightly highermolecular weight

[0058] Purification of EPI-HNE-4.

[0059] 2.5 l of the PEY-53 CM is harvested by centrifugation and thesupernatant is 0.2μ microfiltered as described above. A 30 Kdaultrafiltration is performed on the 0.2μ filtrate, and when theretentate volume is reduced to about 250 ml, a diafiltration of theretentate is performed at a constant retentate volume (250 ml) for 60min at a rate of 10 ml/min. The resulting final volume of 30 Kdafiltrate is about 3 l.

[0060] Properly processed EPI-HNE-4 is purified substantially free ofalternatively-processed form and other fermenter culture components byion exchange chromatography on a polyacrylamide gel carrying sulfonategroups (Macroprep 50S Biorad). A 50 ml bed volume of Macroprep 50S isequilibrated with 10 mM sodium citrate pH 3.5, the 30 Kdaultrafiltration filtrate applied to the column (complete binding ofEPI-HNE-4 to the column is confirmed by demonstrating the absence of hNEinhibitor activity in the column eluate), and the column washed with tencolumn volumes of 10 mm sodium citrate, pH 3.5. The column is theneluted with a gradient from 10 mM ammonium acetate pH3.5 to 1M ammoniumacetate pH3.5 in 30 column volumes. EPI-HNE4 is eluted at the end of thegradient as a single symmetric peak. The eluate is freeze-dried.

[0061] 300 mg of purified active EPI-HNE4 were thus obtained from 2.5 lof CM corresponding to a yield of about 20%.

[0062] SDS-PAGE analysis showed a purity of the product over 95%. Themain contaminant was a green fluorescent pigment.

EXAMPLE 2 Purification of EPI-HNE4 According to the Method of theInvention Wherein Step (b) is a Hydrophobic Interaction ChromatographyStep

[0063] 100 l of the PEY-53 CM obtained as described in Example 1 werecollected and passed over an expanded bed as follows: 10 l ofchromatographic matrix (Streamline SP from Amersham-Pharmacia) isequilibrated in 50 mM ammonium acetate pH 3.5 and fluidized in the samebuffer to 30 l at 300 cm/h. After loading, the column is washed in the10 μM ammonium acetate pH 3.5 to obtain an absorption at 280 nm below0.05. The beads are packed to 10 l and EPI-HNE-4 is recovered by washingthe column in 1 M ammonium acetate pH 4.5 buffer.

[0064] Thus was obtained a 10 l solution containing about 60 g ofproteins and pigments (as determined by spectrometric assay at 280 rmand by Coomassie protein assay). RP-HPLC (silica column Licrosphere.100RP from pharmacia/gradient of water+1% TFA and acetonitrile+1% TFA)showed that the amount of EPI-HNE4 was only about 30 g and thealternatively-processed form was not separated from the main form. Thecontamination by green contaminants is detectable.

[0065] The solution was sterile-filtered on a 22 μm filter (Millipack200 from Millipore) before further purification.

[0066] Hydrophobic interaction chromatography was conducted by passingthe above 10 l solution on a BioProcess (Pharmacia) system, using aphenyl-sepharose Fast Flow matrix from Pharmacia in a 15 l BPTG columnfrom Pharmacia. The buffers used were A: sodium acetate 50 mM pH 4.5+1MNaCl, and B: sodium acetate 50 mM pH 4.5. The elution was performed inone step at 100% B with a flow rate of 300 cm/h.

[0067] The eluate contained about 15 g of purified EPI-HNE4 (asdetermined by spectrometric assay at 280 nm, Coomassie protein assay andbiological activity assay).

[0068] RP-HPLC showed that the alternatively-processed form was notseparated. No green pigment was detectable.

[0069] Cation exchange chromatography was then performed using aBioprocess chromatographic system from Pharmacia. The matrix used wasMacroprep High S matrix from BioRad (rigid matrix based on cross-linkedmethacrylate carrying sulphonate surface groups), in a 15 l BPG200column from Pharmacia. The buffers used were A: ammonium acetate 10 mMpH 3.5, B: sodium acetate 50 mM pH 6.2 and C: 10 mM ammonium bicarbonatepH 7.8. A first elution in buffer B was used to separate thealternatively processed form. Elution was then performed by one step at100% C with a flow rate of 300 cm/h.

[0070] The eluate contained about 12 g of purified EPI-HNE4 (asdetermined by spectrometric assay at 280 nm, Coomassie protein assay andbiological activity assay), corresponding to an overall yield of thepurification process of about 40%.

[0071] RP-HPLC showed less than 1.5% of the alternatively-processedform. No green pigment was detectable.

EXAMPLE 3 Purification of EPI-HNE4 According to the Method of theInvention Wherein Step (b) is a Reverse Chromatography Step

[0072] 100 l of the PEY-53 CM obtained as described in Example 1 werecollected and passed over an expanded bed as follows: 10 l ofchromatographic matrix (Streamline SP from Amersham-Pharmacia) isequilibrated in 50 mM ammonium acetate pH 3.5 and fluidized in the samebuffer to 30 l at 300 cm/h. After loading, the column is washed in the10 mM ammonium acetate pH 3.5 to obtain an absorption at 280 nm below0.05. The beads are packed to 10 l and EPI-HNE-4 is recovered by washingthe column in 1 M ammonium acetate pH 4.5 buffer.

[0073] Thus was obtained a 10 l solution containing about 60 g ofproteins and pigments (as determined by spectrometric assay at 280 nmand Coomassie protein assay). RP-HPLC (silica column Licrosphere 100RPfrom Pharmacia/gradient of water+1% TFA and acetonitrile+1% TFA) showedthat the amount of EPI-HNE4 was about 30 g and thealternatively-processed form was not separated from the correct form.The contamination by green contaminants is detectable.

[0074] The solution was sterile-filtered on a 22 μm filter (Millipack200 from Millipore) before further purification.

[0075] Reverse phase chromatography was conducted by passing the above10 l solution on a BioProcess (Pharmacia) system, using a CG-300 Mmatrix from Tosohaas in a 50 l BTSS column from Pharmacia. The buffersused were A: water+0.1% TFA, and B: acetonitrile+0.1% TFA. The columngradient was 25-45% B in 40 column volume and the flow rate was 450cm/h.

[0076] The eluate contained about 30 g of purified EPI-HNE4 (asdetermined by spectrometric assay at 280 nm, Coomassie protein assay andbiological activity assay).

[0077] RP-HPLC showed less than 2% of the alternatively-processed form.No green pigment was detectable.

[0078] Cation exchange chromatography was then performed using aBioprocess chromatographic system from Pharmacia. The matrix used wasMacroprep High S matrix from BioRad (rigid matrix based on cross-linkedmethacrylate carrying sulphonate surface groups), in a 0.5 l BPG100column from Pharmacia. The buffers used were A: ammonium acetate 10 mMpH 3.5, and B: 10 mM ammonium bicarbonate pH 7.8. Elution was performedby step at 100% B and the flow rate was 300 cm/h.

[0079] The eluate contained about 25 g of purified EPI-HNE4 (asdetermined by spectrometric assay at 280 nm, Coomassie protein assay andbiological activity assay), corresponding to an overall yield of thepurification process of about 40%.

[0080] RP-HPLC showed less than 2% of the alternatively-processed form.No green pigment was detectable.

[0081] Only traces of acetonitrile were present.

[0082] These traces of acetonitrile were completely eliminated either byfreeze-drying or by diafiltration.

EXAMPLE 4 Purification of EPI-HNE4 According to the Method of theInvention Wherein Step (b) is a Reverse Chromatography Step and Step (e)is Used

[0083] 100 l of the PEY-53 CM obtained as described in Example 1 werecollected and passed over an expanded bed as follows: 10 l ofchromatographic matrix (Streamline SP from Amersham-Pharmacia) isequilibrated in 50 mM ammonium acetate pH 3.5 and fluidized in the samebuffer to 30 l at 300 cm/h. After loading, the column is washed in the10 mM ammonium acetate pH 3.5 to obtain an absorption at 280 nm below0.05. The beads are packed to 10 l and EPI-HNE-4 is recovered by washingthe column in 1 M ammonium acetate pH 4.5 buffer.

[0084] Thus was obtained a 10 l solution containing about 60 g ofproteins and pigments (as determined by spectrometric assay at 280 nmand Coomassie protein assay). RP-HPLC (silica column Licrosphere 100RPfrom pharmacia/gradient of water+1% TFA and acetonitrile+1% TFA) showedthat the amount of EPI-HNE4 was about 30 g and thealternatively-processed form is not separated from the correct form. Thecontamination by green contaminants is detectable.

[0085] The solution was sterile-filtered on a 22 μm filter (Millipack200 from Millipore) before further purification.

[0086] Reverse phase chromatography was Conducted by passing the above10 l solution on a BioProcess (Pharmacia) system, using a CG-300 Mmatrix from Tosohaas in a 50 l BTSS column from Pharmacia. The buffersused were A: water+0.1% TFA, and B: acetonitrile+0.1% TFA. The columnwas washed in 25% B to was the green pigments and the unidentifiedcoumpound thus in 60% B to eluate the EPI-hNE-4 protein. The flow ratewas 450 cm/h.

[0087] The eluate contained about 25 g of purified EPI-HNE4 (asdetermined by spectrometric assay at 280 nm, Coomassie protein assay andbiological activity assay).

[0088] RP-HPLC showed that the alternatively-processed form wasconserved. No green pigment was detectable.

[0089] Cation exchange chromatography was then performed using aBioprocess chromatographic system from Pharmacia. The matrix used wasMacroprep High S matrix from BioRad (rigid matrix based on cross-linkedmethacrylate carrying sulphonate surface groups), in a 0.5 l XK50 columnfrom Pharmacia. The buffers used were A: sodium acetate 10 mM pH 2.0,and B: 10 mM sodium acetate pH 2.0+Polyethylene imine 1%. Elution wasperformed by step at 100% B and the flow rate was 300 cm/h.

[0090] The eluate contained about 50 g of purified EPI-HNE4 (asdetermined by spectrometric assay at 280 nm, Coomassie protein assay andbiological activity assay)

[0091] The eluate contained about 50 mg/ml of EPI-hNE-4 at low ionicstrength (15 mS/cm).

[0092] The pH of this eluate was increased to pH 4.5 with sodiumhydroxyde 1 M and allowed to crystallize overnight.

[0093] The crystals were recovered by centrifugation (30 min, 10 000 g)and resuspended in 10 mM ammonium bicarbonate buffer before freezedrying.

[0094] 20 g of EPI-hNE-4 protein were thus recovered, corresponding toan overall yield of the purification process of about 66%.

[0095] RP-HPLC showed less than 5% of the alternatively-processed form.No green pigment nor any material absorbing at a wave length of 200 nmwere detectable.

EXAMPLE 5 Purification of EPI-HNE4 According to the Method of theInvention Wherein Optional Step (b) is Not Used and Step (e) is Used

[0096] This example will highlight the advantage of using step (e).Since purification by crystallisation step (e) is very efficient ineliminating the green pigments, step (b) seems not to be necessary whenstep (e) is used.

[0097] The experimental conditions are the same as in Example 4, exceptfor step (b) which is not used. In step (e), several cycles ofsonication/crystallisation are introduced.

[0098] The amounts of EPI-HNE4 obtained after step (a), step (c) andstep (e) for four runs of fermentation using different volumes offermentation broth are set out in Table 1 below. TABLE 1 VOLUME OFEPI-HNE4 Overall FERMENT- after After After After yield of ATIONfermentation step step step purification BROTH (g) (a) (c) (e) % 1 L 0.30.29 0.32 0.18 60 10 L 3 2.95 2.9 1.2 40 50 L 5* 5 5 2.8 56 100 L 3029.4 29.2 14 46

1. Process for the purification of EPI-HNE proteins, from the culturemedium of a host strain for the expression of said proteins, comprisingthe steps of: (a) passing a derived part of the culture medium over anexpanded bed of cationic exchange adsorbent or a mechanically andchemically inert micromembrane, in order to recover an eluate, (b)optionally conducting chromatographic separation of proteins, accordingto their hydrophobicity, on the resulting eluate, (c) passing theresulting eluate over a cationic exchange column, (d) optionallyfiltering the resulting medium such as to obtain a sterile filtrate. 2.Process according to claim 1 which comprises the further step of (e)causing precipitation of EPI-HNE in a crystallised form and recoveringthe protein crystals
 3. Processing according to any of claims 1 and 2claim 1, wherein step (c) is performed at a pH between 1.8 and 5.0,preferably between 2.0 and 3.0
 4. Process according to claim 3, whereinthe elution in step (c) is performed under conditions such as to obtaina high concentration of EPI-HNE and a low ionic strength.
 5. Processaccording to claim 4, wherein the elution is obtained by displacementchromatography.
 6. Process according to claim 2, wherein in step (e)precipitation of EPI-HNE in a crystallised form is conducted in anaqueous vehicle at a pH between 3.0 and 8.0, preferably between 4.0 and6.0.
 7. Process according to claim 6, wherein the concentration ofEPI-HNE in the aqueous vehicle is from 1 to 80 mg/ml, preferably from 2to 50 mg/ml.
 8. Process according to claim 2, wherein step (e) isperformed using cycles of crystallisation/sonication.
 9. Processaccording to claim 1, wherein step (b) is a hydrophobic interactionchromatography step where the matrix is a polymer carrying a ligand andhaving a hydrohobicity between the hydrophobicity conferred by a C2alkyl group and the hydrophobicity conferred by a C8 alkyl group. 10.Process according to claim 9 wherein the matrix is a polymer carrying aligand selected from the group consisting of ethyl, propyl, isopropyl,butyl, isobutyl, tertiobutyl, pentyl, isopentyl, hexyl, phenyl,isopropanol, isobutanol, C4-C6 ether, preferably phenyl.
 11. Processaccording to claim 1, wherein step (b) is a reverse phase chromatographystep conducted on a matrix composed of synthetic hydrophobic beads. 12.Process according to claim 11, wherein the matrix is apolystyrene-divinylbenzene matrix, preferably with beads or 60-90 μm,more preferably 75 μm, and with a pore size greater than 150 Å,preferably a pore size of 300 Å.